Solution state conformational preferences of dipeptides derived from N-aminoazetidinecarboxylic acid: An assessment of the hydrazino turn

Article abstract of DOI:10.1021/jo400736t

Four model compounds and four dipeptides containing N- aminoazetidinecarboxylic acid (AAzC) and a particular stereoisomer of 2-aminocyclobutanecarboxylic acid (ACBC) were studied to establish their solution state conformational preferences, particularly regarding the ability of AAzC to induce a three-center hydrogen-bonded folding feature known as a "hydrazino turn". On the basis of IR and NMR experiments, supported by molecular modeling, the AAzC residue adopted a trans configuration amenable to the formation of a cyclic eight-membered hydrogen bond conformation in solution, in all cases studied. The implication of the heterocyclic nitrogen atom of AAzC in the trans-like structure was demonstrated via a refined ¹H- ¹⁵N HMBC experiment giving exploitable data at natural ¹⁵N isotopic abundance, providing unprecedented evidence for the solution state hydrazino turn conformation. The predominance of this secondary structural feature depended on the configuration of the neighboring ACBC residue in the dipeptides: while the trans-ACBC derivatives prefer the hydrazino turn, the cis-ACBC derivatives may also populate low-energy 10-membered hydrogen-bonded ring structures. X-ray diffraction analysis of three compounds confirmed the presence of a solid state hydrazino turn in two cases, with geometries similar to those deduced from the solution state studies, but in the third compound, no intramolecular hydrogen-bonding feature was in evidence.

Full text of DOI:10.1021/jo400736t

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Article
Solution State Conformational Preferences of Dipeptides Derived from N-
Aminoazetidinecarboxylic Acid: An Assessment of the Hydrazino Turn
Amandine ALTMAYER-HENZIEN, Valerie DECLERCK, Denis Merlet, Jean-
Pierre BALTAZE, Jonathan FARJON, Régis Guillot, and David J. Aitken
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/jo400736t • Publication Date (Web): 21 May 2013
Downloaded from http://pubs.acs.org on May 21, 2013
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Solution State Conformational Preferences of Dipeptides Derived from
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N-Aminoazetidinecarboxylic Acid: An Assessment of the Hydrazino Turn
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Amandine Altmayer-Henzien,§ Valérie Declerck,§ Denis Merlet,† Jean-Pierre Baltaze,‡
Jonathan Farjon,† Régis Guillot‡ and David J. Aitken*,§
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†
§Laboratoire de Synthèse Organique et Méthodologie, Laboratoire de RMN en Milieu
Orienté, and ‡Services Communs, ICMMO (UMR CNRS8182), Université Paris Sud, 15 Rue
Georges Clemenceau, 91405 Orsay cedex, France
david.aitken@u-psud.fr
Table of Contents
Abstract
Four model compounds and four dipeptides containing N-aminoazetidinecarboxylic acid
(AAzC) and a particular stereoisomer of 2-aminocyclobutanecarboxylic acid (ACBC) were
studied to establish their solution-state conformational preferences, particularly regarding the
ability of AAzC to induce a three-center hydrogen bonded folding feature known as a
“hydrazino turn”. On the basis of IR and NMR experiments, supported by molecular
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modelling, the AAzC residue adopted a trans configuration amenable to the formation of a
cyclic 8-membered hydrogen bond conformation in solution, in all cases studied. The
implication of the heterocyclic nitrogen atom of AAzC in the trans-like structure was
demonstrated via a refined H-15N HMBC experiment giving exploitable data at natural N
isotopic abundance, providing unprecedented evidence for the solution-state hydrazino turn
conformation. The predominance of this secondary structural feature depended on the
configuration of the neighbouring ACBC residue in the dipeptides: while the trans-ACBC
derivatives prefer the hydrazino turn, the cis-ACBC derivatives may also populate low energy
10-membered hydrogen bonded ring structures. X-Ray diffraction analysis of three
compounds confirmed the presence of a solid-state hydrazino turn in two cases, with
geometries similar to those deduced from the solution state studies, but in the third compound
no intramolecular hydrogen bonding feature was in evidence.
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Introduction
The specific functions performed by natural peptides are the result of their well-defined
architectures, which depend on locally-ordered secondary structural features. Significant
efforts have been made to understand and control the factors which determine the
conformational preferences of peptides and a key contemporary challenge at the interface of
chemistry and biology is the rational design of specifically-folded peptidomimetic materials.
A fruitful approach has been to examine the behavior of short oligomer sequences assembled
from monomer building blocks which are analogs of Nature’s amino acids.1-5 In this respect,
peptides which contain β-amino acids are of considerable importance, in part because they
can behave as biologically active peptidomimetics6-8 but also because they have emerged as
self-structuring manifolds for the construction of foldamers.9-13
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In the peptidomimetics area, hydrazino peptides have attracted particular attention. These
compounds are obtained when one or more of the α-amino acids in a peptide sequence are
replaced by an α-hydrazino acid. Conceptually, an α-hydrazino acid is an aza-analog of a β-
amino acid, whose β-carbon has been replaced by a nitrogen atom.14 This strategy for the
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backbone modification of α-peptides has been known for some time,15 and has provided
peptidomimetic materials showing biologically activity16-18 or resistance to proteolysis.19 One
of the specific features of hydrazino peptides is their capacity to adopt an intramolecular 8-
membered ring hydrogen bond, a conformational preference which is also known for
oligomers of α-aminoxy acids.20-23 On the basis of a number of X-ray crystal structures,24-28 it
was suggested that the additional backbone nitrogen in a hydrazino peptide might participate
in a 3-center hydrogen bond, called a hydrazino turn, which stabilizes the 8-membered ring
secondary structure (Figure 1). The significance of the hydrazino turn in solution has been
more difficult to demonstrate, although some linear and cyclic aza-β3-peptides do appear to
have accessible 8-membered ring conformations.19,29-36 Nonetheless, theoretical studies have
not confirmed a decisive role for the 3-center hydrogen bond in determining the folding
properties of hydrazino peptides.37 Only one study of an alternating mixed peptide constructed
from a cyclic β-amino acid (2-aminocyclopentanecarboxylic acid) and its aza-analogue has
been carried out, revealing that the adoption of an 8-ring pseudocyclic network was
configuration-dependent and did not appear to involve the 3-center hydrazino turn.38
Further examination of the conformational behavior of building blocks which combine a
cyclic molecular structure with α-hydrazino acid is thus warranted, and these criteria are met
by N-aminoazetidinecarboxylic acid (AAzC; Figure 1).39,40 This compound is an aza-analogue
of 2-aminocyclobutanecarboxylic acid (ACBC; Figure 1) whose trans configuration is
reported to stabilize 8-membered hydrogen bond ring structures in short peptides41,42 while
the cis configuration forms only intra-residue 6-membered hydrogen bonds.43 In this work, we
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report on the detailed structural analysis of dipeptide derivatives of AAzC, leading to direct
solution state evidence for the bifurcated hydrogen bond of a hydrazino turn which stabilizes
an 8-membered hydrogen bond ring.
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Figure 1. The hydrazinoturn bonding pattern and the molecular structures of AAzC and
ACBC.
Results and discussion
To begin our studies, a model amide of (S)-AAzC was prepared. The Boc-protected hydrazino
acid 1, prepared from 6-azauracil according to the published procedure,39 was condensed with
cyclobutylamine to give amide 2. For comparison purposes, the cyclobutyl ester 3 and the
cyclobutylamides of N-Boc trans- and cis-ACBCs, 6 and 7 respectively, were also prepared.
The ACBC precursors 4 and 5 were readily available following the published protocols.44 The
syntheses of compounds 2, 3, 6 and 7 are outlined in Scheme 1.
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Scheme 1. Preparation of model compound amide 2 and related derivatives 3, 6 and 7.
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Solution state FTIR absorption spectroscopic analysis of amide 2 was carried out in
chloroform at a concentration of 10 mM (Figure 2). The NH stretching frequency domain
generally shows a free amide absorption above 3400 cm–1, whereas this band is shifted to
lower frequencies (between 3350 and 3250 cm–1) when the NH is involved in a hydrogen
bond. The IR absorption spectrum of 2 revealed a stretching band at ca. 3310 cm–1, attributed
to the hydrogen bonded amide NH(10) and a second band at ca. 3360 cm–1, corresponding to
the free carbazate NH(4) with a Z geometry.31 Although the Z conformer is usually less
favoured, the formation of an intramolecular hydrogen bond between C=O(3)⋅⋅⋅H(10)
N
stabilizes this conformer over the E conformer (Scheme 2). This behaviour contrasts with that
of the cyclobutyl ester 3. The solution state IR absorption spectrum of 3 showed a strong
absorption band at ca. 3440 cm–1 and a weak band at ca. 3360 cm–1. In this case, no
intramolecular hydrogen bond involving C=O(3) is possible, so ester 3 exists as a mixture of E
and Z conformers in which the former predominates.
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Figure 2. FTIR absorption spectra of compounds 2, 3, 6 and 7 (10 mM solution in
CHCl3).
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Scheme 2. Equilibria for E and Z conformers of amide 2 and ester 3.
O
O
N
N
H10
H
O
N
OtBu
N
N
N
4
H
H
3
O
OtBu
E-2
Z-2
O
O
O
O
N
OtBu
N
O
N
N
H
H
O
OtBu
E-3
Z-3
In comparison, the absorption spectrum of the reference trans-ACBC amide 6, expected to
adopt the 8-membered hydrogen bond ring, showed a band at ca. 3445 cm–1 corresponding to
the carbamate NH and another band at ca. 3290 cm–1 assigned to the hydrogen bonded amide
NH. In contrast, cis-ACBC amide 7 showed a single absorption band at ca. 3435 cm–1
assigned to both the free amide NH and the hydrogen bonded carbamate NH. The small red
shift (10 cm–1) observed for the carbamate NH suggests that the hydrogen bond interaction is
of only moderate strength. These observations are in agreement with the formation of an 8-
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membered hydrogen bond ring for 6 and a 6-membered hydrogen bond ring for 7 (Figure 3),
which in turn supports the 8-membered hydrogen bond ring conformer proposed for AAzC
amide 2 in the previous paragraph.
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Figure 3. Structures of reference amides 6 and 7.
O
NH
N
H
O
O
H
N
N
H
OtBu
tBuO
O
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NMR spectroscopic analysis of the AAzC amide 2 was carried out in CDCl3 solution.
Complete attribution of all 1H and 13C signals was achieved by means of 1D selective TOCSY
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and 2D HSQC experiments. The H NMR spectrum showed a strongly deshielded signal for
NH(10) (8.46 ppm) suggesting that this proton is implicated in a strong hydrogen bond. The
intramolecular nature of this hydrogen bonding was confirmed by the absence of significant
NH chemical shift variation with concentration in the range 5–60 mM. In an isotopic
exchange experiment (addition of methanol-d4 to a 60 mM solution in CDCl3), only the NH(4)
signal moved to lower field and disappeared rapidly (See Supporting Information). These
results are in good agreement with an 8-membered hydrogen bond ring conformation. A
series of NOESY experiments was performed on amide 2 in order to extract interatomic
distances for application in molecular modeling studies. The main correlations are illustrated
in Figure 4. A hybrid Monte Carlo molecular mechanics (MCMM) conformational search was
carried out on 2 in vacuum using SPARTAN’06 software45 and the MMFF94 force field
without restraints. Four low-energy conformer families emerged and each was subjected to a
DFT geometrical optimization using B3LYP 6-31G** basis sets. After refinement, the four
conformer families were still accessible. Each family of conformers was assessed for its
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compatibility with the NOESY-derived interatomic distances using the DYNAMO molecular
modelling facility in NMR PIPE.46 The lowest energy family correlated perfectly with the
NMR data, and corresponded to an 8-membered hydrogen bond ring with the cyclobutyl
group facing the t-butyl group. The minimum energy conformers are shown in Figure 5 and
important structural parameters are presented in Table 1.
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Figure 4. NOE contacts observed for amide 2.
Figure 5. Lowest energy conformer of amide 2.
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Table 1. Significant calculated parameters for compounds 2 and 12-15 in solution state
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Distances (Å)
H bond
Torsion
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8
Hydrazino turn
10-Membered ring
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H(4)-
H(6’)
2.58
2.59
2.61
2.56
3.66
3.63
2.60
2.62
2.59
H(4)-
H(8’)
2.69
2.67
2.80
2.84
3.53
3.57
2.66
2.71
2.65
H(8)-
H(10)
3.36
3.36
3.34
3.32
3.36
3.35
3.36
3.37
3.39
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C=O(3)⋅⋅⋅H(10)
N
N(5)⋅⋅⋅H(10)
N
C=O(15)⋅⋅⋅H(4)N C=O(15)⋅⋅⋅H(10)
N
(N(4)–N(5))^(C(8)–C(9))
2
2.18
2.15
2.11
2.09
2.29
2.29
2.32
2.33
–96.2°
–96.7°
–96.3°
–96.7°
–91.9°
–92.3°
–96.2°
–96.3°
–97.1°
12
16
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13a
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13c
13d
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2.22
2.50
2.46
2.30
2.14
2.21
2.27
2.26
2.26
2.31
15a
15b
The potential implication of nitrogen N(5) in the stabilization of the 8-membered hydrogen
bond ring in compound 2 was evaluated through a newly developed H-15N heteronuclear
1
NMR experiment. IMPACT HMBC (IMProved and Accelerated Constant-Time
Heteronuclear Multiple-Bond Correlation) NMR spectroscopy is a variation of the 2D HMBC
technique which provides a high quality 2D spectrum with minimal artifacts and enhanced
sensitivity, thus improving the detection of weak interaction such as hydrogen bonds which
involve nuclei (such as 15N) which have low natural abundance.47,48 Firstly, unambiguous
assignment of each of the three 15N NMR signals of amide 2 was achieved via a H-15N
1
HSQC experiment. The IMPACT HMBC experiment was then carried out, and showed a
correlation peak between N(5) and H(10) which was interpreted as the result of a hydrogen bond
between these two atoms. These studies provide convincing evidence for the formation of a
hydrazino turn in solution (Figure 6).
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Figure 6. 1H-15N IMPACT HMBC NMR for compound 2 in CDCl3 with a
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1
superimposition of a part of the H-15N HSQC (in blue) allowing the attribution of the
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different nitrogen atoms at 263 K.
The ensemble of evidence presented above suggests that the AAzC residue presents its own
preferred structural pattern based on a hydrazino turn stabilized 8-membered hydrogen bond.
To establish the stability of this secondary structural feature, we decided to study dipeptides
combining an AAzC unit and each of the stereoisomers of ACBC, which are also known to
possess a pronounced local folding potential.
Four dipeptides combining (S)-AAzC with a designated stereoisomer of ACBC were
prepared. Dipeptides 12-15 were synthesized in good yield by coupling the Boc-hydrazino
acid 1 with the methyl ester of each stereoisomer of ACBC (obtained by deprotection of
compounds 8-11, which in turn were obtained by esterification of the appropriate ACBC
stereoisomer) as shown in Scheme 3. The four dipeptides 12-15 were fully characterized
using the same suite of structural analyses (IR, NMR, molecular modelling) as described
above for the AAzC amide 2.
FTIR analysis of the two dipeptides containing a trans-ACBC residue 12 and 14 gave results
similar to those obtained for the model amide 2, i.e. a stretching band at ca. 3360 cm–1
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assigned to the carbazate NH vibration and a strong band at ca. 3300 cm–1 from the hydrogen
bonded amide NH (Figure 7). Thus, these two dipeptides appear to adopt the cyclic 8-
membered hydrogen bond conformation. The case of the two cis derivatives 13 and 15 is less
clear cut, since three absorption bands are present: a small stretching band at ca. 3435 cm–1
associated to a free amide NH and/or to the carbazate NH vibration of the E conformer, a
second absorption band at ca. 3360 cm–1 assigned to the carbazate NH vibration in the Z
conformation, and a third band at ca. 3300 cm–1 corresponding to the hydrogen bonded amide
NH. Thus, dipeptides 13 and 15 exist as a mixture of conformers. While these observations
can be in part explained by the presence of a hydrazino turn conformer, they also indicate the
presence of a conformation in which the amide NH(10) is non-hydrogen bonded or in which
NH(4) and C=O(3) are not involved in a hydrogen bond, leaving the carbazate function to adopt
its preferred E geometry. While this additional conformer is of interest, it was not possible at
this stage to identify it unambiguously. If a longer-distance intramolecular non-covalent
interaction were implicated, such as a 10-membered ring, the carbazate moiety could be in
either a E or Z geometry but NH(4) would be hydrogen-bonded, giving an absorption at ca.
3300 cm–1, while the non-bonded NH(10) would appear at ca. 3440 cm–1. On the other hand, an
extended conformation would imply a free NH(4) and an “unrestrained” carbazate geometry
(i.e. E > Z), as long as a non-bonded NH(10) giving rise to an intense absorption signal at 3440
cm–1 and a low absorption at 3360 cm–1.
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Figure 7. FTIR absorption spectra of dipeptides 12-15 (10 mM solution in CHCl3).
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Scheme 3. Preparation of dipeptides 12-15.
1
After complete attribution of all H and 13C signals, 1D NMR experiments (concentration
dependence, isotopic exchange) showed that dipeptides 12-15 behaved in a similar fashion to
the model amide 2: a low-field, non concentration-dependent chemical shift for NH(10) and an
isotopic exchange sensitive NH(4) signal again indicated a cyclic 8-membered hydrogen bond
conformer in all four cases.
NOESY experiments on dipeptides 12-15 provided correlation patterns similar to that
observed for the model amide 2 and were used to extract interatomic distances (See
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Supporting information). A MCMM conformational search followed by DFT geometry
optimization was carried out as described above for amide 2 (Data are collected in Table 1).
For the two dipeptides containing a trans-ACBC residue, 12 and 14, the conformational
search gave respectively two and three low-energy conformer families which remained after
DFT geometry optimization. In both cases, the lowest potential energy corresponded to the
hydrazino turn conformation and fitted perfectly with the NOESY NMR data (Figure 8). The
situation was more complex for the two cis-ACBC peptides 13 and 15. The conformational
search gave nine and eight families of conformers, respectively. Conformational refinement
reduced the number of accessible families of conformers to six and five, respectively. The
large number of conformer families is partly due to “family twins” arising from the two
conformations that a cis-ACBC unit can adopt, each with one substituent pseudo-axial and the
other pseudo-equatorial. For dipeptide 15, the two hydrazino turn conformations 15a and 15b
emerged as the lowest energy families, and appear to be populated equally. In the case of
dipeptide 13, the hydrazino turn was relinquished in favor of a 10-membered hydrogen bond
ring in the two lowest energy conformers, 13c and 13d, although this observation alone does
not explain the FTIR and 1D NMR isotopic exchange data described above. The four low-
energy conformer families were therefore subjected to an ab initio geometrical optimization at
the B3LYP 6-31G** level of theory in chloroform (implicit method) using GAUSSIAN’09
software.49 The four low energy conformer families - two hydrazino turn families 13a and
13b and two 10-membered hydrogen bond ring families 13c and 13d - emerged with very
similar energies suggesting roughly equal populations. This situation best explains the FTIR
results, and is consistent with the NOESY NMR data.
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Figure 8. Lowest energy conformers of compounds 12-15.
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The four dipeptides 12-15 were analysed by the 1H-15N IMPACT HMBC NMR experiment in
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CDCl3 to search for an interaction between NH(10) and N(5). The sensitivity of all H-15N
1
correlations was enhanced by a factor in the range 2 to 8 compared to the standard H-15N
HMBC experiment, and a specific correlation between NH(10) and N(5) was observed in all
four cases. These observations provide convincing evidence for the presence of the bifurcated
hydrogen bonding system of a hydrazino turn in solution.
We were able to deduce the coupling constants (h1JNH) through the hydrogen bond between
NH(10) and N(5) then use the values to evaluate the distance between the two atoms. For each
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peptide 12-15, a deconvolution procedure allowed the comparison of a reconstructed H
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spectrum with the extracted rows from the H-15N IMPACT HMBC spectra.50 The
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reconstructed spectra which showed the best fit with the extracted spectra provided the value
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h1
of the JNH coupling constant. These coupling constants were then applied in an empirical
equation which had previously been developed to assess the distance between an amide NH
and an imidazole N in proteins.51 In the event, the h1JNH coupling constants for dipeptides 12-
15 were in the range 2.8 to 3.4 Hz, which corresponds to a N(5)⋅⋅⋅H(10) distance of 2.8-2.9 Å.
h1
The same analysis conducted on the model amide 2 provided a JNH value of 3.2 Hz which
corresponds to a N(5)⋅⋅⋅H(10) distance of 2.9 Å. The interatomic distances deduced by this
method are slightly higher than those obtained from the molecular modelling studies (Table
1), but still constitute a reasonable approximation considering that the equation used is purely
empirical.
While the principle objective of this work was to study AAzC derivatives in solution state, it
was instructive to examine and compare the results with solid state data when available.
Single crystals of model AAzC amide 2 and dipeptide 14 were amenable to X-ray diffraction
analysis and the crystal structure conformations of these compounds are shown in Figure 9.
Each compound adopts a conformation very similar to its lowest-energy solution state
structure with a hydrazino turn evidently contributing to the 8-membered hydrogen bond ring.
In both cases, the AAzC unit adopts a trans-like configuration, with a backbone torsion angle
of –97.3° for 2 and –93.9° for 14. This latter value is slightly lower than those calculated from
solution state studies (Table 1) which might be the result of packing effects in the solid state.
In each case, H(10) participates in a three-center hydrogen bond with C=O(3) and N(5) for which
distances are in the range 2.2-2.3 Å (Table 2).
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Figure 9. X-Ray structures of amide 2 and dipeptide 14.
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Table 2. Significant parameters for crystal structure of compounds 2 and 14.
Distances (Å)
H(4)-H(8)
2.66
H bond
Torsion
H(4)-H(6’)
2.51
H(8)-H(10)
3.20
C=O(3)⋅⋅⋅H(10)
2.22
N
N(5)⋅⋅⋅H(10)
2.25
N
(N(4)–N(5))^(C(8)–C(9))
–97.3°
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14
2.48
2.62
3.22
2.29
2.29
–93.9°
Intriguingly, single crystal analysis of dipeptide 15 showed an extended molecular structure in
which no intramolecular interactions were present (Figure 10). The off-set head-to-head
alignment of the molecules in the crystal facilitates the formation of a network of
intermolecular C=O(9)⋅⋅⋅H(4)’N hydrogen bonds. N(5) is not involved in any non-covalent
interaction.
Figure 10. X-Ray structure of dipeptide 15.
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This first detailed structural study of AAzC derivatives in solution indicates that the title
compound conveniently combines the rigidity of a cyclic molecular skeleton with a hydrazino
acid function in order to facilitate the formation of a hydrazino turn conformer in small
peptides. NMR data, in particular, provided strong evidence for a non-covalent interaction
between the AAzC ring-nitrogen and the amide NH of the adjacent residue, consistent with
the tenet that a bifurcated hydrogen bond is a central feature of the cyclic 8-membered
hydrazino turn feature. Nonetheless, low energy conformations structured around a cyclic 10-
membered ring hydrogen bond also appears accessible, which suggests that AAzC might be
able to adapt its local structure to accommodate the demands of neighboring residues. AAzC
thus emerges as a potentially useful building block for inclusion in peptides with predictable
yet malleable conformational behavior.
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Experimental section
All reagents and solvents were of commercial grade and were used without further
purification. Dichloromethane was dried over activated alumina and THF was distilled from
sodium/benzophenone. Flash chromatography was performed with SDS silica gel (35-70 μm).
Analytical thin-layer chromatography was performed with 0.25 mm commercial silica gel
plates (EMD, Silica Gel 60F254). TLC plates were visualised by UV fluorescence at 254 nm
then revealed using a phosphomolybdic acid solution (10% in EtOH) or a ninhydrin solution
(0.3% in n-BuOH). Retention factors (Rf) are given for such analyses. Routine nuclear
magnetic resonance (NMR) data were acquired on a spectrometer operating at 360 MHz for
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1H and at 90 MHz for C. Chemical shifts (δ) are reported in ppm from tetramethylsilane.
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Splitting patterns for H NMR signals are designated as: s (singlet), d (doublet), t (triplet), q
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(quadruplet), quint (quintuplet), bs (broad singlet) or m (multiplet). Coupling constants (nJ)
are reported in Hz. High resolution mass spectrometry (HRMS) data were recorded using the
electrospray ionization technique in positive mode (ESI+) with a tandem Q-TOF analyzer.
Samples for Fourier-transform infrared spectroscopy (IR) were prepared as solution in CHCl3
(10 mM) in a NaCl solution cell. Maximum absorbances (ν) are given for significant bands in
cm-1. Melting points were obtained in open capillary tubes and are uncorrected. Optical
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T
rotations were measured using a 10 cm quartz cell. Values for [α]D were obtained with the D-
line of sodium at the indicated temperature T, using solutions of concentration (c) in units of
g·100 mL–1.
General procedure for the preparation of cyclobutylamides derivatives. To an ice cold
solution of Boc-hydrazino acid 1 or Boc-amino acid 4 or 5 (1.0 mmol, 1 eq.) in dry CH2Cl2
(20 mL) were added 1-hydroxybenzotriazole monohydrate (189.2 mg, 1.4 mmol, 1.4 eq.) and
cyclobutylamine (94 μL, 1.1 mmol, 1.1 eq.). The mixture was stirred for 10 min at 0 °C then
1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (287.6 mg, 1.5 mmol, 1.5 eq.)
was added. The resulting mixture was then stirred for 14 h at room temperature, then washed
successively with 1 M aqueous KHSO4 (10 mL) and saturated NaHCO3 (10 mL). The organic
layer was dried over MgSO4, filtered and evaporated under reduced pressure. Flash
chromatography (CH2Cl2/EtOAc) gave the cyclobutylamide derivative 2, 6 or 7.
(S)-N-Cyclobutyl-1-(t-butyloxycarbonylamino)azetidine-2-carboxamide (2). Coupling of
Boc-(S)-AAzC 1 with cyclobutylamine according to the general procedure followed by flash
chromatography (CH2Cl2/AcOEt = 50/50) gave the cyclobutylamide (–)-2 (216.2 mg, 80%) as
a white solid. Mp 190.5-191.1 °C (dec.); Rf 0.22 (CH2Cl2/AcOEt = 50/50); [α]D24 −53 (c 0.50,
CHCl3); IR (CHCl3) ν 3359, 3310, 3020, 3006, 2980, 2940, 2896, 2873, 1714, 1652, 1544,
1
1497; H NMR (360 MHz, CDCl3) δ 1.43 (s, 9H), 1.58-1.78 (m, 2H), 1.86-2.10 (m, 3H),
2
3
2.13-2.38 (m, 3H), 3.32 (pseudo dd, 1H, J = 15.7 Hz, J = 8.8 Hz), 3.61-3.68 (m, 1H), 3.88
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(pseudo t, 1H, J = 8.8 Hz), 4.30-4.43 (m, 1H), 6.15 (bs, 1H), 8.46 (bs, 1H); C NMR (90
MHz, CDCl3) δ 15.3, 19.8, 28.3, 30.7, 31.0, 44.2, 54.7, 72.2, 81.1, 155.6, 170.4; HRMS
(ESI+) Calcd for C13H23N3NaO3 [M+Na]+: 292.1632, found 292.1622.
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N-Cyclobutyl-(1R,2R)-2-(t-butyloxycarbonylamino)cyclobutanecarboxamide
(6).
Coupling of Boc-(1R,2R)-ACBC 4 with cyclobutylamine according to the general procedure
followed by flash chromatography (CH2Cl2/AcOEt = 70/30) gave the cyclobutylamide (–)-6
(219.9 mg, 82%) as a white solid. Mp 225-226 °C (dec.); Rf 0.26 (CH2Cl2/AcOEt = 70/30);
23
[α]D −9 (c 0.50, CHCl3); IR (CHCl3) ν 3446, 3290, 3019, 2981, 2938, 2897, 2874, 1694,
1
1644, 1554, 1501; H NMR (360 MHz, CDCl3) δ 1.46 (s, 9H), 1.60-1.79 (m, 3H), 1.80-2.03
2
3
(m, 3H), 2.03-2.22 (m, 2H), 2.22-2.39 (m, 2H), 2.85 (pseudo dd, 1H, J = 18.2 Hz, J = 9.2
Hz), 4.05-4.17 (m, 1H), 4.28-4.44 (m, 1H), 4.93 (bs, 1H), 8.17 (bs, 1H); 13C NMR (90 MHz,
CDCl3) δ 15.4, 18.7, 24.9, 28.5, 30.9, 31.2, 44.7, 48.8, 50.4, 80.6, 156.4, 172.0; HRMS
(ESI+) Calcd for C14H24N2NaO3 [M+Na]+: 291.1679, found 291.1667.
N-Cyclobutyl-(1R,2S)-2-(t-butyloxycarbonylamino)cyclobutanecarboxamide
(7).
Coupling of (1R,2S)-ACBC 5 with cyclobutylamine according to the general procedure
followed by flash chromatography (CH2Cl2/AcOEt = 80/20) gave the cyclobutylamide (–)-7
(253.5 mg, 94%) as a white solid. Mp 174-175 °C (dec.); Rf 0.50 (CH2Cl2/AcOEt = 70/30);
[α]D24 −108 (c 0.50, CHCl3); IR (CHCl3) ν 3436, 3021, 2980, 2951, 2897, 1699, 1657, 1502;
1H NMR (360 MHz, CDCl3) δ 1.40 (s, 9H), 1.63-1.74 (m, 2H), 1.75-1.91 (m, 3H), 2.01-2.11
(m, 1H), 2.13-2.37 (m, 4H), 3.11 (pseudo dt, 1H, 3J = 8.2 Hz, 3J = 2.6 Hz), 3.29-3.42 (m, 2H),
5.29 (bs, 1H), 5.73 (bs, 1H); 13C NMR (90 MHz, CDCl3) δ 15.3, 18.3, 25.5, 29.2, 31.2, 31.4,
44.9, 46.5, 79.4, 155.4, 171.9; HRMS (ESI+) Calcd for C14H24N2NaO3 [M+Na]+: 291.1679,
found 291.1674.
Cyclobutyl (S)-1-(t-butyloxycarbonylamino)azetidine-2-carboxylate (3). To a solution of
Boc-(S)-AAzC 1 (46.5 mg, 0.22 mmol, 1 eq.) in CH2Cl2 (2.5 mL) were added successively
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DMAP (39.4 mg, 0.32 mmol, 1.5 eq.) and cyclobutanol (19 μL, 0.24 mmol, 1.1 eq.). The
mixture was then cooled to 0 °C, and EDCI·HCl (61.8 mg, 0.32 mmol, 1.1 eq.) was added.
The solution was stirred for 5 min at 0 °C then 15 h at room temperature, at which point the
solvent was evaporated under reduced pressure. Flash chromatography (petroleum
ether/EtOAc = 90/10) gave the methyl ester (–)-3 (34.3 mg, 59%) as a colorless oil. Rf 0.25
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(petroleum ether/EtOAc = 80/20); [α]D −110 (c 0.50, CHCl3); IR (CHCl3) ν 3438, 3359,
3019, 2984, 2891, 1728, 1717, 1477, 1467; 1H NMR (360 MHz, CDCl3) δ 1.42 (s, 9H), 1.51-
1.66 (m, 1H), 1.70-1.83 (m, 1H), 1.92-2.15 (m, 3H), 2.16-2.38 (m, 3H), 3.54 (ddd, 1H, 2J =
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14.1 Hz, J = 5.9 Hz, J = 4.5 Hz ), 3.99 (pseudo dd, 1H, J = 14.1 Hz, J = 7.7 Hz), 4.72
(pseudo t, 1H, 3J = 7.5 Hz), 5.01 (pseudo quint., 1H, 3J = 7.6 Hz), 6.36 (bs, 1H); 13C NMR (90
MHz, CDCl3) δ 13.5, 18.6, 28.4, 30.3, 53.8, 66.4, 69.2, 80.4, 154.8, 171.8; HRMS (ESI+)
Calcd for C13H22N2NaO4 [M+Na]+: 293.1472, found 293.1457.
General procedure for the preparation of methyl esters. To a solution of Boc-amino acid 4
or 5 (215.3 mg, 1.0 mmol, 1 eq.) in CH2Cl2 (11 mL) were added successively DMAP (12.2
mg, 0.1 mmol, 0.1 eq.) and MeOH (0.13 mL, 13 mmol, 13 eq.). The mixture was then cooled
to 0 °C, and EDCI·HCl (210.9 mg, 1.1 mmol, 1.1 eq.) was added. The solution was stirred for
5 min at 0 °C then 15 h at room temperature, at which point the solvent was evaporated under
reduced pressure. Flash chromatography (petroleum ether/EtOAc = 90/10) gave the methyl
ester 8-11.
Methyl (1R,2R)-2-(t-butyloxycarbonylamino)cyclobutanecarboxylate (8). Coupling of
Boc-(1R,2R)-ACBC 4 with methanol according to the general procedure followed by flash
chromatography (Petroleum Ether/AcOEt = 90/10) gave the methyl ester (–)-8 (192.6 mg,
84%) as a white solid. Rf 0.32 (Petroleum Ether/AcOEt = 80/20); [α]D19 −53 (c 1.08, CHCl3);
1H NMR (360 MHz, CDCl3) δ 1.37 (s, 9H), 1.78-1.96 (m, 3H), 2.11-2.25 (m, 1H), 2.87-3.06
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(m, 1H), 3.63 (s, 3H), 4.08-4.24 (m, 1H), 5.02 (bs, 1H); C NMR (90 MHz, CDCl3) δ 18.2,
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27.3, 28.4, 46.8, 48.9, 51.7, 79.5, 154.7, 173.5.
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Methyl (1R,2S)-2-(t-butyloxycarbonylamino)cyclobutanecarboxylate (9). Coupling of
Boc-(1R,2S)-ACBC 5 with methanol according to the general procedure followed by flash
chromatography (Petroleum Ether/AcOEt = 90/10) gave the methyl ester (–)-9 (174.4 mg,
79%) as a white solid. Rf 0.44 (Petroleum Ether/AcOEt = 80/20); [α]D20 −130 (c 1.09, CHCl3);
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1H NMR (360 MHz, CDCl3) δ 1.38 (s, 9H), 1.84-2.03 (m, 2H), 2.11-2.37 (m, 2H), 3.29-3.40
13
(m, 1H), 3.66 (s, 3H), 4.43-4.49 (m, 1H), 5.33 (bs, 1H); C NMR (90 MHz, CDCl3) δ 18.5,
28.4, 29.5, 45.3, 45.9, 51.7, 79.4, 154.8, 174.7.
Methyl (1S,2S)-2-(t-butyloxycarbonylamino)cyclobutanecarboxylate (10). Coupling of
Boc-(1S,2S)-ACBC ent-4 with methanol according to the general procedure followed by flash
chromatography (Petroleum Ether/AcOEt = 90/10) gave the methyl ester (+)-10 (190.3 mg,
83%) as a white solid. Rf 0.32 (Petroleum Ether/AcOEt = 80/20); [α]D18 +58 (c 1.08, CHCl3);
1H NMR (360 MHz, CDCl3) δ 1.38 (s, 9H), 1.41 (s, 9H), 1.81-2.00 (m, 3H), 2.17-2.28 (m,
13
1H), 2.89-3.05 (m, 1H), 3.67 (s, 3H), 4.13-4.26 (m, 1H), 4.84 (bs, 1H); C NMR (90 MHz,
CDCl3) δ 18.3, 27.4, 28.4, 47.0, 49.0, 51.8, 79.7, 154.8, 173.5.
Methyl (1S,2R)-2-(t-butyloxycarbonylamino)cyclobutanecarboxylate (11). Coupling of
Boc-(1S,2R)-ACBC ent-5 with methanol according to the general procedure followed by flash
chromatography (Petroleum Ether/AcOEt = 90/10) gave the methyl ester (+)-11 (167.4 mg,
73%) as a white solid. Rf 0.44 (Petroleum Ether/AcOEt = 80/20); [α]D20 +124 (c 1.09, CHCl3);
1H NMR (360 MHz, CDCl3) δ 1.37 (s, 9H), 1.83-2.02 (m, 2H), 2.10-2.38 (m, 2H), 3.28-3.40
13
(m, 1H), 3.65 (s, 3H), 4.32-4.49 (m, 1H), 5.34 (bs, 1H); C NMR (90 MHz, CDCl3) δ 18.5,
28.4, 29.5, 45.3, 45.9, 51.7, 79.4, 154.8, 174.7.
General procedure for the preparation of dipeptides. TFA (0.69 mL, 9 mmol, 30 eq.) was
added dropwise to a solution of Boc-amino ester 8-11 (68.8 mg, 0.3 mmol, 1 eq.) in CH2Cl2
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(2.1 mL). The mixture was stirred for 30 min at room temperature, then the solvent and the
excess TFA were evaporated under reduced pressure to give an oil (c. 110 mg). The resulting
TFA salt TFA·H2N-ACBC-OMe was used directly in the next reaction.
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To a cold (–10 °C) solution of Boc-(S)-AAzC 1 (64.9 mg, 0.3 mmol, 1 eq.) in dry THF (0.3
mL) were added successively Et3N (132 μL, 0.95 mmol, 3.2 eq.) and IBCF (39 μL, 0.3 mmol,
1 eq.). The solution was stirred for 5 min at –10 °C, then a solution of TFA·H2N-ACBC-
OMe in dry THF (0.3 mL) was added. Residual salts were taken up by rinsing with dry THF
(0.3 mL) and added to the cold reaction mixture. The resulting mixture was stirred for 30 min
at –10 °C and then for 3 h at room temperature, at which point the solution was filtered and
evaporated under reduced pressure. Flash chromatography (CH2Cl2/EtOAc = 50/50) of the
residue gave the dipeptide 12-15.
(1R,2R)-Methyl
2-((S)-1-(t-butyloxycarbonylamino)azetidine-2-
carboxamido)cyclobutane carboxylate (12). Coupling of Boc-(S)-AAzC 1 with TFA·H2N-
(1R,2R)-t-ACBC-OMe according to the general procedure followed by flash chromatography
(CH2Cl2/AcOEt = 50/50) gave the dipeptide (–)-12 (87.9 mg, 90%) as a white solid. Mp 110
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°C; Rf 0.16 (CH2Cl2/AcOEt = 50/50); [α]D −82 (c 0.50, CHCl3); IR (CHCl3) ν 3359, 3301,
3019, 2982, 2870, 1724, 1661, 1544, 1498; 1H NMR (360 MHz, CDCl3) δ 1.40 (s, 9H), 1.82-
2.06 (m, 4H), 2.11-2.28 (m, 2H), 3.09-3.20 (m, 1H), 3.34 (pseudo dd, 1H, 2J = 15.9 Hz, 3J =
8.5 Hz), 3.56-3.72 (m, 1H), 3.64 (s, 3H), 3.86 (pseudo t, 1H, 3J = 8.8 Hz), 4.51 (pseudo quint,
1H, 3J = 8.3 Hz), 6.32 (bs, 1H), 8.67 (bs, 1H); 13C NMR (90 MHz, CDCl3) δ 18.7, 19.8, 26.8,
28.3, 46.4, 46.7, 51.8, 54.5, 72.0, 81.0, 155.7, 170.7, 173.5; HRMS (ESI+) Calcd for
C15H25N3NaO5 [M+Na]+: 350.1686, found 350.1685.
(1R,2S)-Methyl
2-((S)-1-(t-butyloxycarbonylamino)azetidine-2-
carboxamido)cyclobutane carboxylate (13). Coupling of Boc-(S)-AAzC 1 with TFA·H2N-
(1R,2S)-c-ACBC-OMe according to the general procedure followed by flash chromatography
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(CH2Cl2/AcOEt = 50/50) gave the dipeptide (–)-13 (90.4 mg, 92%) as a white solid. Mp 153
°C; Rf 0.15 (CH2Cl2/AcOEt = 50/50); [α]D25 −166 (c 0.50, CHCl3); IR (CHCl3) ν 3430, 3360,
3299, 3012, 2979, 2894, 1723, 1661, 1527, 1501; 1H NMR (360 MHz, CDCl3) δ 1.43 (s, 9H),
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1.82-2.01 (m, 2H), 2.11-2.40 (m, 4H), 3.37 (pseudo dd, 1H, J = 11.7 Hz, J = 8.2 Hz), 3.51
2
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(pseudo dd, 1H, J = 15.6 Hz, J = 8.6 Hz), 3.56-3.64 (m, 1H), 3.58 (s, 3H), 4.07 (pseudo t,
1H, 3J = 8.8 Hz), 4.62 (pseudo quint, 1H, 3J = 8.2 Hz), 6.22 (bs, 1H), 8.30 (bs, 1H); 13C NMR
(90 MHz, CDCl3) δ 19.2, 19.7, 27.7, 28.4, 44.6, 45.3, 51.5, 54.3, 70.4, 81.0, 155.3, 171.5,
173.4; HRMS (ESI+) Calcd for C15H25N3NaO5 [M+Na]+: 350.1686, found 350.1679.
(1S,2S)-Methyl 2-((S)-1-(t-butyloxycarbonylamino)azetidine-2-carboxamido)cyclobutane
carboxylate (14). Coupling of Boc-(S)-AAzC 1 with TFA·H2N-(1S,2S)-t-ACBC-OMe
according to the general procedure followed by flash chromatography (CH2Cl2/AcOEt =
50/50) gave the dipeptide (+)-14 (69.7 mg, 71%) as a white solid. Mp 120 °C; Rf 0.17
24
(CH2Cl2/AcOEt = 50/50); [α]D +4 (c 0.50, CHCl3); IR (CHCl3) ν 3360, 3301, 3020, 2981,
2955, 2934, 2871, 1725, 1661, 1542, 1497; 1H NMR (360 MHz, CDCl3) δ 1.36 (s, 9H), 1.81-
2.02 (m, 4H), 2.11-2.27 (m, 2H), 2.98-3.08 (m, 1H), 3.35 (pseudo dd, 1H, 2J = 15.7 Hz, 3J =
8.8 Hz), 3.55 (s, 3H), 3.53-3.62 (m, 1H), 3.88 (pseudo t, 1H, 3J = 8.8 Hz), 4.47 (pseudo quint,
1H, 3J = 8.1 Hz), 6.68 (bs, 1H), 8.71 (bs, 1H); 13C NMR (90 MHz, CDCl3) δ 18.8, 19.6, 27.1,
28.2, 46.0, 46.6, 51.6, 54.3, 71.7, 80.8, 155.8, 170.9, 173.5; HRMS (ESI+) Calcd for
C15H25N3NaO5 [M+Na]+: 350.1686, found 350.1694.
(1S,2R)-Methyl
2-((S)-1-(t-butyloxycarbonylamino)azetidine-2-
carboxamido)cyclobutane carboxylate (15). Coupling of Boc-(S)-AAzC 1 with TFA·H2N-
(1S,2R)-c-ACBC-OMe according to the general procedure followed by flash chromatography
(CH2Cl2/AcOEt = 50/50) gave the dipeptide (+)-15 (77.7 mg, 79%) as a white solid. Mp 109
26
°C; Rf 0.19 (CH2Cl2/AcOEt = 50/50); [α]D +40 (c 0.50, CHCl3); IR (CHCl3) ν 3439, 3359,
3295, 3019, 2979, 2955, 2895, 2871, 1726, 1660, 1531, 1496; 1H NMR (360 MHz, CDCl3) δ
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1.39 (s, 9H), 1.76-2.00 (m, 2H), 2.13-2.41 (m, 4H), 3.34-3.45 (m, 2H), 3.55-3.64 (m, 1H),
3.72 (s, 3H), 3.94 (pseudo t, 1H, 3J = 8.7 Hz), 4.68 (pseudo quint, 1H, 3J = 8.3 Hz), 6.15 (bs,
1H), 8.59 (bs, 1H); 13C NMR (90 MHz, CDCl3) δ 18.7, 19.9, 27.7, 28.3, 44.6, 45.3, 51.7,
54.5, 71.5, 80.7, 155.1, 171.1, 173.3; HRMS (ESI+) Calcd for C15H25N3NaO5 [M+Na]+:
350.1686, found 350.1690.
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Acknowledgments
A. A.-H. is the beneficiary of an Allocation Spécifique pour Normalien doctoral research
grant from the French Ministry of Higher Education and Research. We thank COST (Action
CM 0803) for facilitating constructive networking in relation to this work.
Supporting Information
1
1
General information, copies of H and 13C NMR spectra for all new compounds, H-15N
IMPACT HMBC NMR spectra and X-ray diffraction data for compounds 2, 14 and 15 in cif
format. This material is available free of charge via the Internet at http://pubs.acs.org.
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